Identification of 1,3,6,8-Tetrahydroxynaphthalene Synthase (ThnA) from Nocardia sp. CS682

Type III polyketide synthase (PKS) found in bacteria is known as 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). Microbial type III PKSs synthesize various compounds that possess crucial biological functions and significant pharmaceutical activities. Based on our sequence analysis, we have identified a putative type III polyketide synthase from Nocardia sp. CS682 was named as ThnA. The role of ThnA, in Nocardia sp. CS682 during the biosynthesis of 1,3,6,8 tetrahydroxynaphthalene (THN), which is the key intermediate of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) was characterized. ThnA utilized five molecules of malonyl-CoA as a starter substrate to generate the polyketide 1,3,6,8-tetrahydroxynaphthalene, which could spontaneously be oxidized to the red flaviolin compound 2,5,7-trihydroxy-1,4-naphthoquinone. The amino acid sequence alignment of ThnA revealed similarities with a previously identified type III PKS and identified Cys138, Phe188, His270, and Asn303 as four highly conserved active site amino acid residues, as found in other known polyketide synthases. In this study, we report the heterologous expression of the type III polyketide synthase thnA in S. lividan TK24 and the identification of THN production in a mutant strain. We also compared the transcription level of thnA in S. lividan TK24 and S. lividan pIBR25-thnA and found that thnA was only transcribed in the mutant.

UV-protective compound known as IBR-3, which is formed by removing the complete PKS region of nargenicin [15]. Different enzymes like methyltransferases and glycosyltransferase are involved in the post-modification of 1,3,6,8-tetrahydroxynaphthalene (THN) during the formation of IBR-3 ( Fig. 2) [15,21,22]. Due to the crucial role played by ThnA in forming the core THN scaffold, we carried out a sequence alignment of ThnA with established type III PKSs. This alignment yielded a high level of bootstrap support, thereby strengthening the case for heterologous expression.
In this study, we characterized the function of thnA by heterologous expression in S. lividan sp. TK24. ThnA is a key enzyme in the production of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene from five malonyl-CoA molecules to form THN, which undergoes further modifications by methyltransferases and glycosyltransferase enzymes involved in this pathway to yield the final product IBR-3.

Bacterial Strains, Plasmids, and Culture Conditions
S. lividans TK24 was utilized as a host for heterologous expression and cultured in 50 ml of R2YE medium, which is composed of 0.02% potassium sulfate, 10.3% sucrose, 1% magnesium chloride, 0.5% yeast extract, 0.01% disfco casamino acid, and 1% glucose. To extract genomic DNA, Nocardia sp. CS682 was cultured for 5 days at  37°C and 200 rpm in brain heart infusion (BHI) medium [23]. The bacterial strains E. coli XL1 and E. coli ET12567 were cultured at 37°C on either solid or liquid Luria Bertani (LB) medium. The cloning vector utilized in this study was pGEM-T Easy (Promega, USA). Recombinant DNA was constructed with the plasmid vector pIBR25 (Fig.  S1A), which was placed under the regulation of an ermE promoter to facilitate the expression of DNA in Streptomyces [24]. In order to transform the recombinant DNA into the protoplasts of S. lividans TK24, the standard protocol utilized in our previous study was followed [25]. Standard commercial sources were utilized to purchase chemicals and reagents for biochemical and molecular biology analyses.

Construction of Recombinants and Transformation into Streptomyces lividans TK24
The TIANamp bacterial DNA kit was used to isolate and purify genomic DNA from Nocardia sp. CS682 [21]. Polymerase chain reaction (PCR) was used to amplify the thnA (1,125-bp) from Nocardia sp. CS682 genomic DNA using the following primer pair: 5-GGATCCATGGCAATCTTGTGTCGAC-3 (forward, BamHI) and 5-GAATTCTCATAGGACTACCTCCCCT-3 (reverse EcoRI) with high fidelity pfu DNA polymerase (Takara, Japan). PCR analysis was performed on a thermal cycler from Takara, Japan, using the following conditions: a 7 min initial denaturation at 95°C, 30 cycles of denaturation at 92°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min. The final step involved a 7 min extension at 72°C. To prepare the PCR products for further analysis, they were purified from a 0.7% agarose gel, and then ligated into the pGEM ® -T Easy vector from Promega, Wisconsin, USA. The BamHI-EcoRI fragment containing thnA was ligated into pIBR25 to construct pIBR25-thnA (Fig. S1). pIBR25-thnA was transferred into E. coli XL1-Blue and the plasmids were isolated and transformed into the final E. coli ET-12567 construct to obtain demethylated plasmid for transformation in S. lividans TK24 by protoplast transformation.

Protoplast Preparation, Transformation in S. lividansp TK24
S. lividans TK24 protoplast preparation was performed as described previously [25]. Briefly, 150 μl of protoplasts were treated for 12 min with 15 μl of 0.1 mM aurintricarboxylic acid (ATA) (Sigma-Aldrich, USA) and mixed with 20 μl of plasmid DNA for 2 min. After that, a 40% (w/v) polyethylene glycol 1000 (PEG) solution from Merck-Schuchardt, Germany, was added to the mixture (200 μl) and gently mixed. The solution was then briefly centrifuged to remove PEG and the resulting sample was resuspended in protoplast buffer [26]. The transformed protoplasts were spread onto R2YE regeneration plates at 28°C for 16 h. Then, an overlay of 0.3% agar solution containing 15 μg/ml thiostrepton was added, and the plates were screened on R2YE plates with the same concentration of thiostrepton (15 μg/ml). A parallel transformation and screening were also performed using pIBR25 to generate S. lividans pIBR25.

Extraction, Isolation, and In Vivo Analysis
S. lividans TK24 and other mutants (S. lividans pIBR25-thaA, S. lividans pIBR25) were cultivated in 50 ml of R2YE media supplemented with 15 μg/ml of thiostrepton at a temperature of 28°C for a duration of 5 days and extracted with a double volume of ethyl acetate. The organic layer was then concentrated using a rotary evaporator and the resulting concentrated sample was dissolved in a suitable amount of methanol, filtered, and subsequently examined through reverse-phase high-performance liquid chromatography (HPLC, Mightysil RP-18 GP 250-4.6, Japan), and mass analysis was performed by liquid chromatography-electron spray ionization/mass spectrometry (LC-ESI/MS) in the positive ion mode using an Acquity column (UPLC; Waters Corp., USA) coupled with a SYNAPT G2-S column (Waters Corp.). The sample was eluted using a solvent mobile phase gradient of 0.1% trifluoroacetic acid (TFA) in water and 100% acetonitrile (ACN) over a period of 0 to 12 min at a temperature of 35°C. The volume of the injected sample was 10 microliters.

RNA Sample Preparation and Reverse Transcription PCR Analysis
To extract total RNA, each 5 ml aliquot of culture that was grown for approximately 72 h was suspended in RNA protect Bacteria Reagent (Qiagen, Germany) for a duration of 5 min. RNA isolation was carried out using the RNeasy Mini kit (Qiagen) in accordance with the guidelines provided by the manufacturer. DNase (Qiagen) was used to treat contaminating DNA in the RNA samples, and the lack of contamination was verified by PCR analysis using the RNA as a template. To assess the purity and concentration of the total RNA, a spectrophotometer (Shimadzu, UV-1601 PC) was utilized to measure the optical density at 260/280 nm. Reverse transcription PCR (RT-PCR) was performed with a QuantiTech SYBR Green RT-PCR kit (Qiagen). The primers used for thnA (158 bp) were 5-CCGAGCGCTAGGAAACG-3 (forward) and 5-ATCCGAGCTGCGCGATA-3 (reverse), and for the control housekeeping gene [rpoB (181 bp)] they were 5-AGTTCGGCGAGTACGAGTC-3 (forward) and 5-ACCTTGGCGAGGTCGTAG-3 (reverse). For the RT-PCR analysis, identical amounts of RNA from each sample were utilized. The reaction conditions involved initial cDNA synthesis at 50°C for a duration of 30 min, followed by initial denaturation at 98°C for 10 min and 45 cycles. Each cycle consisted of denaturation at 98°C for 30 sec, annealing at 55−68°C for 30 sec, and elongation at 72°C for 1 min. The amplified DNA samples were further subjected to 1-2% agarose gel electrophoresis to verify the amplification product. To verify that the amplified products were not originating from chromosomal DNA that might have contaminated the RNA preparations, negative controls were conducted using Taq DNA polymerase without reverse transcriptase. To determine whether thnA was expressed in S. lividans TK24, we cultured the wild-type, i.e., S. lividans TK24, and thnA overexpressed in S. lividans pIBR25-thnA in HT medium. Samples were taken on the third day, and mRNA was isolated using a standard protocol. RpoB is a housekeeping gene in S. lividans TK24 and was used as a control.

Sequence and Phylogenetic Analysis of ThnA
The genomic analysis of Nocardia sp. CS682 [21] showed that the biosynthetic gene cluster (BGC) responsible for the synthesis of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene consisted of PKS (ThnA) and three O-methyltransferases (ThnM1, ThnM2, and ThnM3) (Fig. 2). ThnA was putatively annotated for the production of 1,3,6,8-tetrahydroxynaphtalene (THN) from malonyl-CoA. The thnA gene encodes a protein consisting of 374 amino acids (AA), and the gene's overall G+C content is 56%. According to BLAST searches, thnA shows significant similarity with other type III PKS genes in the database. The amino acid sequence alignment of ThnA, as illustrated in Fig. 3, exhibited the existence of four extensively conserved residues, namely Cys 138 , Phe 188 , His 270 , and Asn 303 , which are present in other known PKSs. From the structure and functional studies of chalcone synthase and other PKSs, Cys 138 serves as a nucleophile that starts the reaction and makes the enzyme-bound polyketide intermediate [7,27,28]. Phe 188 provides a hydrophobic environment that promotes the decarboxylation of malonyl-CoA [29], ionic interaction of His 270 plays a crucial role in stabilizing the thiolate anion of Cys 168 [30], and Asn 303 is responsible for catalyzing the decarboxylation of malonyl-CoA [1,30]. Additionally, it has been previously demonstrated that Tyr 224 assists in substrate specificity, and its aromatic ring is considered essential for ThnA to recognize malonyl-CoA as a starter unit, much like the two other aromatic amino acids, Trp and Phe [29]. The amino acid alignment and conserved domains in ThnA suggest that it could serve as a THNS. A phylogenetic tree was constructed using the protein sequences of ThnA and their closest homologs (Fig. S2). Phylogenetic tree analysis of ThnA revealed that it had the closest relationships with functionally characterized PKS.

Heterologous Expression of thnA
In this study, S. lividans TK24 was utilized as a host for heterologous expression. The construction of pIBR25-thnA involved cloning the DNA fragment containing thnA into pIBR25, which was subsequently transformed into S. lividans TK24 via protoplast transformation using a standard protocol [25]. Confirmation of successful transfer of the plasmid into the heterologous host was achieved through plasmid isolation and restriction digestions (data not shown). S. lividans and S. lividans pIBR25-thnA were cultivated in R2YE liquid medium for a duration of 5 days at a temperature of 28°C. To extract the cultures, a double volume of ethyl acetate was employed, and the resulting organic supernatant fraction was subsequently concentrated using a rotary evaporator. After concentration, the sample was dissolved in a suitable volume of methanol for subsequent HPLC analysis at a UV wavelength of 330 nm by Thermo HPLC series 1100 with a Thermo-C 18 column (5 μm, 4.6 mm × 250 mm). The HPLC mobile phase was composed of solvent A (water with 0.1% trifluoroacetic acid [TFA]) and solvent B (acetonitrile) and followed a linear gradient (10% acetonitrile at 0 min, 10 to 90% B for 1-25 min, 90 to 50% B for 25−28 min, and from 50 to 10% B for 28−30 min), at a constant flow rate of 1 ml/min. The HPLC profile showed a distinct peak at a retention time of (t R ) 15.7 min from S. lividans pIBR25-thnA, but no peak was observed from wild-type or S. lividans pIBR25 (Fig. 4A). The identity of THN and flaviolin (an auto-oxidized product of THN) was further supported by LC-ESI/MS analysis ([M+H] + ion at m/z = 193.0489 found, calcd for C 10

RNA Isolation and Real-Time PCR Analysis
For transcriptional analysis of thnA, S. lividans TK24 and S. lividans pIBR25-thnA were cultured in 50 ml of HT medium. Samples were taken on the third day, and mRNA was isolated using a standard protocol. Gene-specific primers were used, and rpoB, the housekeeping gene in S. lividans, was used as a control. The RT-PCR results showed the band of thnA (158 bp) in S. lividans pIBR25-thnA, which did not exist in the wild-type strain, i.e., S. lividans TK24, whereas the housekeeping rpoB showed a band (181 bp) in both strains (Fig. 5). The amplified sequenced was further confirmed through sequencing after ligation into the pGEM ® -T Easy vector.

Conclusion
In this study, we successfully identified and characterized a type III polyketide synthase (ThnA) from Nocardia CS682. The enzyme was found to use five molecules of malonyl-CoA to synthesize 1,3,6,8 tetrahydroxynaphthalene (THN), which was then modified to form the final product 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene (IBR-3) with UV-protective properties. To validate its function, we performed the heterologous expression of thnA in S. Lividans TK24 and confirmed its expression through RT-PCR. In vivo production of flaviolin showed that the enzyme acted as a THN synthase. This characterization provides insight into the biosynthetic pathways of the important compound IBR-3.